Anti-retroviral treatment using growth hormone

ABSTRACT

The subject invention pertains to methods of treating HIV-1 infected subject comprising the administration of growth hormone to a subject infected by HIV-1. The subject may be undergoing co-administration of anti-retroviral therapies (ARTs), may be untreated with anti-retroviral drugs (ARDs) or may be in a period of time where no ART is being administered. Growth hormone may be administered at a fixed dosage for a set period of time or may be administered at a first dosage for a first period of time that may then be increased or decreased to a second dosage for a second period of time.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/445,501, filed Jan. 12, 2017, the disclosure of which is herebyincorporated by reference in its entirety, including all figures, tablesand amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jan. 5, 2018 and is 2 KB. The entire content of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Anti-retroviral therapy (ART) with anti-retroviral drugs (ARD) hasdramatically reduced the death rate from AIDS and improved the qualityof life of many HIV-infected individuals [1]. A sterilizing cure, inwhich the virus is completely eradicated, would require the eliminationof all replication-competent viruses throughout the body. An alternativeapproach provides for providing an individual with good long-term healthin the absence of ART, or a disease “remission”. Such a “remission” maybe achieved by reducing the amount of residual HIV during ART (the“viral reservoir”) to levels which the immune system can effectivelycontrol [3]. This is commonly referred to as a “functional cure” inwhich the viral reservoir is naturally controlled by the host. Bothforms of cure (sterilizing and functional) would require eliminating, orat least reducing, the reservoirs of HIV infection. Several strategiesaimed at reducing the size of the viral reservoir have been testedwithin the past 5 years. Among them, the “shock and kill” approachconsists in forcing viral gene expression in order to flush out thelatent reservoir. Disappointingly, none of these interventions to datehave had a demonstrable effect on the size of the latent HIV reservoir.Thus, novel strategies are needed in order to improve the reduction ofthe latent HIV reservoir size.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention relates to the treatment ofHIV-infected individuals with growth hormone (GH) (the use of GH for thetreatment of HIV-infected individuals). In certain embodiments of thisaspect of the invention, the HIV-infected individuals are undergoingconcomitant ART (simultaneous treatment with anti-retroviral drugs)(ARD). The present invention also provides other aspects that providevarious GH dosing regimens suitable for the treatment of HIV-infectedindividuals.

Another aspect of the invention is directed to the use of growth hormonefor the reduction of latent HIV reservoir size in HIV-infectedindividuals (e.g., the use of GH to reduce the latent HIV reservoir sizein HIV-infected individuals). Thus, methods of treating HIV-infectedindividuals with growth hormone in amounts effective to reduce thelatent HIV reservoir within the treated individual are provided. Whilethe treatment is contemplated for use in all HIV-infected populations,certain populations may be excluded from treatment within the context ofthe present invention. For example, individuals receiving growth hormonetreatment for HIV-associated wasting (cachexia) can be excluded from thepatient population being treated with growth hormone.

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.The transitional terms/phrases (and any grammatical variations thereof)“comprising”, “comprises”, “comprise”, “consisting essentially of”,“consists essentially of”, “consisting” and “consists” can be usedinterchangeably.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed. In the context ofcompositions containing amounts of GH where the terms “about” or“approximately” are used, these compositions contain the stated amountof the ingredient with a variation (error range) of 0-10% around thevalue (X±10%). When used in the context of time (e.g., days or weeks),the terms “about” or “approximately” provide for variations of up to 5days.

The term “growth hormone (GH)”, as used herein, is intended to includegrowth hormone from various sources. In particular embodiments, the GHis human origin (for example, isolated from biological fluids orobtained by DNA recombinant techniques from prokaryotic or eukaryotichost cells. In a particularly preferred embodiment, GH that isadministered in accordance with the disclosed methods is SEROSTIM®and/or SAIZEN®. SAIZEN and SEROSTIM are somatropin, a recombinant humangrowth hormone (rhGH) produced by genetically engineered mammalian cells(mouse C127). Somatropin is a single-chain, non-glycosylated protein of191 amino acids with two disulfide bridges. Of course other recombinantgrowth hormones can be used in accordance with the claimed invention andnon-limiting examples of such recombinant growth hormones includeNUTROPIN (Genentech), HUMATROPE (Lilly), GENOTROPfN (Pfizer),NORDITROPIN (Novo), and OMNITROPE (Sandoz). Within the context of thisapplication, GH can be administered systemically, and preferablysubcutaneously or intramuscularly. Intradermal, transdermal (e.g. inslow release formulations), intravenous, oral, topical, rectal, andintranasal routes of administering GH to a subject are alsocontemplated.

The terms “co-administration,” “administered in combination with,” andtheir grammatical equivalents encompass administration of two or moreagents to a subject so that both agents and/or their metabolites arepresent in the subject at the same time. Co-administration includessimultaneous administration in separate compositions, administration atdifferent times in separate compositions, or administration in acomposition in which both agents are present.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both pre-clinical humantherapeutics and veterinary applications. In some embodiments, thesubject is a mammal (such as a primate model of disease), and in someembodiments, the subject is human. The terms “subject”, “individual” and“patient” can be used interchangeably.

The terms “simultaneous” or “simultaneously” as applied to administeringagents to a subject refer to administering one or more agents at thesame time, or at two different time points that are separated by no morethan 1 hour. The term “sequentially” refers to administering more thanone agent at two different time points that are separated by more than 1hour, e.g., about 2 hours, about 5 hours, 8 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days or even longer.

ARD that can be used in ART are identified in Table 1. ART may beadministered to an individual treated in accordance with the claimedinvention for a period of time prior to treatment with growth hormone inamounts sufficient to reduce the latent HIV reservoir within thesubject. In certain embodiments, the HIV-infected individual is treatedwith ART for a period of at least 6 months, at least 12 months, at least18 months or at least 24 months prior to treatment with growth hormoneas disclosed herein. The terms “individual”, “subject” and HIV-1infected individual” (and grammatical variants thereof) can be usedinterchangeably within this disclosure.

In the present disclosure, ranges are stated in shorthand, so as toavoid having to set out at length and describe each and every valuewithin the range. Any appropriate value within the range can beselected, where appropriate, as the upper value, lower value, or theterminus of the range. For example, a range of 0.1-1.0 represents theterminal values of 0.1 and 1.0, as well as the intermediate values of0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate rangesencompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.Values having at least two significant digits within a range areenvisioned, for example, a range of 5-10 indicates all the valuesbetween 5.0 and 10.0 as well as between 5.00 and 10.00 including theterminal values. A range can be denoted numerically (e.g., 0.1-1.0) orby the use of the phrase “up to” (e.g., up to 5 days). Where the phrase“up to” is used, the term includes, as a lower limit, the value of 0(zero).

“A week” refers to a period of time of about 5, about 6 or about 7 days.“A month” refers to a period of time of about 28, about 29, about 30 orabout 31 days.

As discussed above, one aspect of the present invention is directed tothe use of growth hormone for the treatment of HIV-infected individuals.In certain embodiments of this aspect of the invention, a reduction oflatent HIV reservoir size in HIV-infected individuals may also beobserved in HIV-infected individuals undergoing treatment according tothe regimens provided herein. Thus, HIV-infected individuals can betreated with growth hormone in amounts sufficient to reduce the latentHIV reservoir within the individual. Individuals who are to be treatedin the context of this invention may be undergoing concomitant(simultaneous) anti-retroviral therapy (ART), may be untreated (noprevious treatment with ARDs) or may be in a period of time during whichART is not, presently, being administered (an “ART-free period”).

While a HIV-infected individual can be treated in an ongoing manner withgrowth hormone, certain embodiments provide for limiting the time thesubject is treated. For example the subject can be treated for a totalof about 52 weeks, about 48 weeks, about 44 weeks, about 40 weeks, about36 weeks, about 32 weeks, about 28 weeks, about 24 weeks, about 20weeks, about 16 weeks, about 12 weeks, about 8 weeks or about 4 weeks.Treatment can be suspended for a period of time, for example a period ofabout 4 weeks to about 52 weeks and then re-initiated at the same doseof GH, and increased dose of GH or a reduced dose of GH. The GH dose canrange between about 1 mg/day to about 5 mg/day and, in preferredembodiments will be about 3 mg/day (30-40 μg/kg/d). Certain aspects ofthe invention provide a dosing regimen that utilizes two or moredifferent dosages of growth hormone for two or more different periods oftime.

One such dosing regimen provides for the administration of growthhormone to an HIV-infected individual in an amount of about 2 mg/day toabout 5 mg/day for a period of about 24 weeks followed by a dosereduction to between about 1 mg/day to about 4 mg/day. In certainpreferred embodiments, the HIV-infected individual is treated with aninitial dose of about 3 mg/day followed by a reduced dose of about 1.5mg/day. In an even more preferred embodiment, the HIV-infectedindividual is treated with an initial dose of about 3 mg/day for aperiod of about 24 weeks followed by a treatment with a reduced dose ofabout 1.5 mg/day for a period of about 24 weeks.

Another dosing regimen provides for the administration of growth hormoneto an HIV-infected individual in an amount of about 2 mg/day to about 5mg/day for a period of about 2 to 52 weeks followed by a dose reductionto between about 1 mg/day to about 4 mg/day for a period of about 2 toabout 52 weeks. In certain preferred embodiments, the HIV-infectedindividual is treated with an initial dose of about 3 mg/day followed bya reduced dose of about 1.5 mg/day. In an even more preferredembodiment, the HIV-infected individual is treated with an initial doseof about 3 mg/day for a period of about 20 weeks to about 30 weeksfollowed by a treatment with a reduced dose of about 1.5 mg/day for aperiod of about 20 weeks to about 30 weeks.

The terms “dose reduction” or “reduced dose” mean that the amountsadministered within the second period of time is reduced as compared tothe amount of growth hormone administered during the first period oftime. The dose reduction can be between at least 20% and 80%. Thus, ifthe HIV-infected individual is treated with an initial dose of 5 mg/day,the reduced dose administered in the second period of time could bebetween 1 mg/day and 4 mg/day. As would be apparent, the reduced doseadministered during the second time period is always less than the doseadministered during the first period of time.

The terms “dose increase” or “increased dose” mean that the amountsadministered within the second period of time is increased as comparedto the amount of growth hormone administered during the first period oftime. The dose increase can be between at least 1.5× and 5×. Thus, ifthe HIV-infected individual is treated with an initial dose of 5 mg/day,the increased dose administered in the second period of time could bebetween 7.5 mg/day and 25 mg/day. As would be apparent, the increaseddose administered during the second time period is always greater thanthe dose administered during the first period of time.

One such dosing regimen provides for the administration of growthhormone to an HIV-infected individual in an amount of about 2 mg/day toabout 5 mg/day for a period of about 24 weeks followed by a doseincrease to between about 3 mg/day to about 25 mg/day. In certainpreferred embodiments, the HIV-infected individual is treated with aninitial dose of about 3 mg/day followed by an increased dose of about 5mg/day. In an even more preferred embodiment, the HIV-infectedindividual is treated with an initial dose of about 3 mg/day for aperiod of about 24 weeks followed by a treatment with an increased doseof about 5 mg/day for a period of about 24 weeks.

Another dosing regimen provides for the administration of growth hormoneto an HIV-infected individual in an amount of about 2 mg/day to about 5mg/day for a period of about 2 to 52 weeks followed by a dose increaseto between about 3.5 mg/day to about 25 mg/day for a period of about 2to about 52 weeks. In certain preferred embodiments, the HIV-infectedindividual is treated with an initial dose of about 3 mg/day followed byan increased dose of about 15 mg/day. In an even more preferredembodiment, the HIV-infected individual is treated with an initial doseof about 3 mg/day for a period of about 20 weeks to about 30 weeksfollowed by a treatment with an increased dose of about 5 mg/day for aperiod of about 20 weeks to about 30 weeks.

HIV-infected individuals who can be treated within the context of thisinvention include those with both detectable and undetectable HIV-1 RNAlevels. In preferred embodiments, subjects with HIV-1 RNA levels belowthe limit of quantification using an FDA-approved assay for a period ofat least 6 month, at least 12 months, at least 18 months or at least 24months prior the initiation of the growth hormone treatment are treatedin accordance with the disclosed methods. Table 2 identified currentlyFDA-approved assays.

As discussed above, the present invention provides methods of reducingthe latent HIV reservoir in an HIV-infected individual. Thus, thedisclosed invention can further comprise testing treated individuals todetermine the level of the HIV reservoir before and/or after treatmentwith GH. The size of the replication competent HIV reservoir can bemeasured by any means known in the art. For example, a quantitativeviral outgrowth assay (mQVOA) can be used to measure the competent HIVreservoir in an individual at the onset of treatment or prior to theonset of treatment and at later times during the treatment regimen (forexample, after 24 and 48 weeks of treatment with recombinant humangrowth hormone). Other markers of viral persistence (HIV DNA and TILDA)can also be measured according to methods known in the art.

The treatment regimens disclosed also provides certain embodimentsvarying the length of time that the subject is treated. For example thesubject can be treated for a total of about 52 weeks, about 48 weeks,about 44 weeks, about 40 weeks, about 36 weeks, about 32 weeks, about 28weeks, about 24 weeks, about 20 weeks, about 16 weeks, about 12 weeks,about 8 weeks or about 4 weeks. As discussed above, certain aspects ofthe invention provide a dosing regimen that utilizes two or moredifferent dosages of growth hormone for two or more different periods oftime.

TABLE 1 Anti-Retroviral Drugs Nucleoside/Nucleotide Analogues AbacavirDidanosine Emtricitabine Lamivudine Stavudine Tenofovir disoproxilfumarate Tenofovir alafenamide Zalcitabine Zidovudine NonnucleosideReverse Transcriptase Inhibitors Delavirdine Efavirenz EtravirineNevirapine Rilpivirine Protease Inhibitors Amprenavir AtazanavirDarunavir Fosamprenavir Indinavir Lopinavir/Ritonavir NelfinavirRitonavir Saquinavir Tipranavir Fusion Inhibitors Enfuvirtide ChemokineCoreceptor Antagonists Maraviroc Integrase Inhibitors DolutegravirElvitegravir Raltegravir Pharmacokinetic Enhancers Cobicistat

TABLE 2 FDA-Approved Quantitative HIV-1 RNA Assays for Viral LoadMonitoring Lower LOQ Upper LOQ Test Name Manufacturer Method (copies/mL)(copies/mL) Abbott RealTime HIV-1 Abbott Real-time  40* 10,000,000Laboratories PCR Cobas AmpliPrep/Cobas TaqMan Roche Real-time 2010,000,000 HIV-1 Test, version 2.0 Diagnostics PCR Cobas HIV-1quantitative NAT Roche Real-time 20 10,000,000 for use on Cobas6800/8800 Diagnostics PCR systems Cobas TaqMan HIV-1 Test, v2.0 RocheReal-time 34 10,000,000 For Use With The High Pure Diagnostics PCRSystem LOQ, limit of quantification. *This lower LOQ applies when 1.0 mLof plasma is used. When 0.5 mL and 0.2 mL of plasma are used, the lowerLOQ is 75 copies/mL and 150 copies/mL, respectively.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.Following are examples which illustrate procedures for practicing theinvention.

These examples should not be construed as limiting. All percentages areby weight and all solvent mixture proportions are by volume unlessotherwise noted. It should also be understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and the scope of the appended claims. Inaddition, any elements or limitations of any invention or embodimentthereof disclosed herein can be combined with any and/or all otherelements or limitations (individually or in any combination) or anyother invention or embodiment thereof disclosed herein, and all suchcombinations are contemplated within the scope of the invention withoutlimitation thereto.

Example 1—HIV-1 Treatment Protocol

HIV-1-infected adults will be enrolled in a prospective, open-label,single-arm study of recombinant human growth hormone treatment.Participants will be treated with recombinant human growth hormone for atotal of 48 weeks.

The initial recombinant human growth hormone dose will be 3 mg/day(30-40 μg/kg/d) for 24 weeks administered by subcutaneous injection onan outpatient basis, followed by dose reduction to 1.5 mg/day for thefinal 24 weeks of the treatment period, also conducted on an outpatientbasis. Participants will be instructed to subcutaneously injectrecombinant human growth hormone between 7 and 10 p.m., daily.

The size of the replication competent reservoir will be measured by amodified version of the quantitative viral outgrowth assay (mQVOA) atbaseline (two measurements prior to the start of treatment with GH), andafter 24 and 48 weeks of treatment with recombinant human growthhormone. Other markers of viral persistence (HIV DNA and TILDA) will bemeasured at various time points during the course of the study.

1. Primary Endpoint: Replication competent HIV between baseline and 48weeks recombinant human growth hormone administration by QVOA.

2. Secondary Endpoint: Integrated HIV DNA between baseline and 48 weeksrecombinant human growth hormone administration by alu-PCR.

3. Secondary Endpoint: Inducible HIV RNA between baseline and 48 weeksrecombinant human growth hormone administration by TILDA.

For inclusion in the study, all of the following criteria must befulfilled:

-   -   1. HIV-1 infection and able to provide written consent.    -   2. Men and women age ≥18 and <40 years.    -   3. Currently on continuous ART for at least 24 months with no        change in regimen in 12 weeks prior to study entry. Some        modifications of ART doses during the 12 weeks prior to study        entry are permitted. In addition, the change in formulation        (e.g., from standard formulation to fixed-dose combination) is        allowed within 12 weeks prior to study entry. A within class        single drug substitution (e.g., switch from tenofovir to        abacavir or raltegravir to dolutegravir) is allowed within 12        weeks prior to study entry.    -   4. CD4+ T-cell count ≥350 cells/mm³ obtained within 30 days        prior to study entry.    -   5. HIV-1 RNA level below the limit of quantification using an        FDA-approved assay for at least 24 months prior to study entry        and confirmed within 60 days prior to study entry. Single        determinations that are between the assay quantification limit        and 200 copies/mL are allowed as long as the preceding and        subsequent determinations are below the level of quantification.    -   6. A female, may be eligible to enter and participate in the        study if she:    -   a. is of non-child-bearing potential defined as physically        incapable of becoming pregnant with documented tubal ligation,        hysterectomy or bilateral oophorectomy or,    -   b. is of child-bearing potential with a negative pregnancy test        at both Screening and Day 1 and agrees to use one of the        following methods of contraception to avoid pregnancy:        -   Complete abstinence from penile-vaginal intercourse from 2            weeks prior to administration of IP, throughout the study,            and for at least 2 weeks after discontinuation of all study            medications;        -   Double barrier method (male condom/spermicide, male            condom/diaphragm, diaphragm/spermicide); barrier methods            must be in use at least 14 days prior to study drug            administration.        -   Any intrauterine device (IUD) with published data showing            that the expected failure rate is <1% per year (not all IUDs            meet this criterion. IUD must be in use at least 30 days            prior to first study drug administration.        -   Male partner sterilization confirmed prior to the female            subject's entry into the study, and this male is the sole            partner for that subject; the vasectomy must be completed 3            months prior to first study drug administration or in the            alternative that a 0 sperm count will suffice.        -   Approved hormonal contraception        -   Any other method with published data showing that the            expected failure rate is <1% per year.            Any contraception method must be used consistently, in            accordance with the approved product label and for at least            2 weeks after discontinuation of recombinant human growth            hormone.

Subjects will be excluded from this study if they fulfill any of thefollowing exclusion criteria:

-   -   1. The following laboratory values obtained at screening:    -   a. Fasting glucose ≥100 mg/dL    -   b. Hemoglobin A1c ≥5.7%    -   c. ALT [serum glutamic pyruvic transaminase (SGPT)]>2 times        upper limit of normal (ULN)    -   d. AST [serum glutamic oxaloacetic transaminase (SGOT)]>2×ULN    -   e. Estimated creatinine clearance ≤50 mL/min by Cockcroft-Gault    -   f. Hemoglobin <11.5 g/dL    -   g. Platelets <100,000/mm³    -   h. ANC <1000/mm³    -   2. Any active or past history of malignancy, except for        localized cutaneous Kaposi's sarcoma (fewer than 10 lesions,        none of which are larger than 2 cm, and not on active therapy).    -   3. Prior therapy with growth hormone or tesamorelin during 12        months preceding screening visit.    -   4. Unstable or untreated hypertension, defined as ≥160/90 mm Hg        at the time of the screening visit.    -   5. History of pancreatitis, carpal tunnel syndrome (unless        resolved by surgical release), diabetes mellitus, angina        pectoris, coronary artery disease, or any disorder associated        with moderate to severe edema (e.g. ascites, nephrotic syndrome,        congestive heart failure, lymphedema).    -   6. Acute or serious illness requiring systemic treatment and/or        hospitalization within 90 days prior to study entry.    -   7. Receipt of antibiotic therapy within 30 days prior to study        entry.    -   8. Chronic hepatitis C infection defined as a positive hepatitis        C antibody and positive hepatitis C RNA at any time prior to        study entry. Subjects who are positive for hepatitis C antibody        but who are HCV RNA negative are permitted in the study.    -   9. Use of immunomodulators (e.g., interleukins, interferons,        cyclosporine), HIV vaccine, systemic cytotoxic chemotherapy, or        investigational therapy within 30 days prior to study entry or        during study.    -   10. Known allergy/sensitivity or any hypersensitivity to        components of study drug or their formulation.    -   11. Recent vaccination within 30 days prior to study entry or        expected vaccination after screening but before baseline visit.    -   12. Active drug or alcohol use or dependence that, in the        opinion of the site investigator, would interfere with adherence        to study requirements.    -   13. Receive testosterone therapy for hypogonadism unless prior        testosterone deficiency is documented.    -   14. Drug or hormone use as follows:    -   a. Men: change in regimen or supraphysiological dose of        testosterone (measured by elevated free testosterone above        normal levels) within 2 months prior to screening;    -   b. anabolic steroids, GH, GH secretagogue, GHRF products or        analogs, IGF-1, or IGF binding protein 3 (IGFBP-3) within 6        months prior to screening;    -   15. Women who are lactating.

Somatropin is a human growth hormone (hGH) produced by recombinant DNAtechnology. Somatropin has 191 amino acid residues and a molecularweight of 22,125 daltons. Its amino acid sequence and structure areidentical to the dominant form of human pituitary growth hormone.Somatropin is produced by a mammalian cell line (mouse C127) that hasbeen modified by the addition of the hGH gene. Somatropin is secreteddirectly through the cell membrane into the cell-culture medium forcollection and purification.

Serostim® is somatropin produced by EMD Serono. Somatropin is a sterilelyophilized powder intended for subcutaneous injection afterreconstitution to its liquid form.

Vials of Serostim® [somatropin (rDNA origin) for injection] approved inCanada (DIN number 02239046) contain either 4 mg, 5 mg, 6 mg, or 8.8 mgof somatropin. Only the 5 mg single-use vial will be used for thisstudy.

Each vial contains the following:

Component Amount Somatropin  5 mg Phosphoric acid 1.2 mg SodiumHydroxide 0.7 mg Sucrose 34.2 mg 

Each Serostim® 5 mg single-use vial is supplied in a combination packagewith Sterile Water for Injection, USP. The pH is adjusted with sodiumhydroxide of phosphoric acid to give a pH of 6.5 to 8.5 afterreconstitution. Study medication will be labeled as an “investigationalproduct to be used only by a qualified investigator”.

Before reconstitution: Vials of somatropin and diluent should be storedat room temperature, (15°−30° C./59°−86° F.). Expiration dates arestated on product labels. After reconstitution: After reconstitutionwith Sterile Water for Injection, USP, the reconstituted solution shouldbe administered immediately (within 3 hours). Although not recommended,it may be stored for up to 24 hours at 2-8° C. As there is nopreservative in this reconstituted solution, any unused solution shouldbe discarded once the dose is given. Avoid freezing reconstituted vialsof somatropin.

All participants will be on combination antiretroviral therapy duringthe entire duration of the study. If a stable regimen was established atleast 2 months prior to screening, testosterone replacement therapy atphysiological doses is acceptable during this study as soon as freetestosterone levels remain in the normal range.

A subject may be using lipid lowering therapy during participation inthis study if he or she has been on a stable dose since at least 2months prior to screening. A subject may be using antihypertensivetherapy during participation in this study if he or she has been on astable dose since at least 1 month prior to screening and has reached asystolic pressure of ≤160 mm Hg and diastolic pressure of ≤90 mm Hg. Anymedications (other than those excluded) that are considered necessaryfor the subject's welfare and will not interfere with the study drug maybe given at the Investigator's discretion.

Clinical Evaluations

Once a candidate for screening has been identified, study details willbe carefully discussed with the subject. The subject will be asked toread and sign the approved informed consent form prior to anyassessments being performed.

Medical history, documented HIV test results, demographic information,date of initiation of highly active antiretroviral therapy, CD4 countnadir, history of antiretroviral treatment and co-infections: hepatitisA, B, C; syphilis, co-morbidities: diabetes, cardiovascular disease,hypercholesterolemia, bone disease, cancers, AIDS related diagnoses andother medications will be obtained at the screening visit. A completephysical exam, including vital signs will also be performed at thescreening visit. A brief physical exam, including vital signs, will beperformed as per site local practices at subsequent visits. In addition,the participants will be asked to bring all the recombinant human growthhormone vials (used and unused) with them to each clinic visit. Thiswill allow to measure adherence and ensure that the recommended dose wasused.

Blood pressure, weight, and height, medication review and adverse eventswill be documented in electronic Case Report Forms (eCRFs) at each visitas indicated in Table 2 (Schedule of Events).

Laboratory Evaluations and Specimen Collection

Blood samples will be obtained for clinical laboratory evaluations ateach study visit as per routine clinical care (see Clinical LaboratoryTests, below). Subjects should be fasting for at least 8 hours prior toblood work. All clinical laboratory evaluations will be performed atsite local laboratories as per local standards of care. When possible,laboratory results will be transferred directly to the main web-baseddatabase by the research assistant. If this is not possible, CRF will beavailable for the site to transcribe the results.

Clinical Laboratory Tests

Hematology Serum Chemistry Serology Other Hgb A1C CMP (Comprehensivemetabolic HCV HIV RNA CBC w/differential panel): Albumin, Alk P'Tase,HBsAg HCV RNA CD4 panel Total, ALT (SGPT), AST Pregnancy test (SGOT),Urea Nitrogen, Calcium, Chloride, C02, Creatinine, glucose, Globulin,Potassium, sodium, Total Bilirubin, Total Protein) Lipid profile (total,HDL/LDL cholesterol, and triglycerides) IGF-1 and IGFBP-3 Quicki index(fasting insulin and glucose)

In addition to the blood collected for the clinical tests describedabove, blood will be collected to measure changes in the size of the HIVreservoir using 3 different methods described below.

Quantitative viral outgrowth assay (QVOA) is recognized as the goldstandard assay for determining the frequency of CD4+ T cells harboringreplication competent proviruses, despite its limitations (timeconsuming, labor-intensive, large blood requirement, expensive, lack ofprecision, and probably incomplete sensitivity for replicationcompetence). A slightly modified a new protocol (modified QVOA, mQVOA)using the MOLT-4/CCR5 cell line, reducing the time of culture and, byeliminating the low donor-dependent efficiency of prolonged co-culture,this assay enhances sensitivity and precision in patients with thesmallest reported reservoirs. The increased sensitivity and speed of theassay is facilitated by 96-well magnetic bead extraction of supernatant(up to 500 μl), which is more efficient than other methods and thendirect transfer of all extracted RNA into a 96-well ultrasensitivesemi-nested real time RT-PCR with a detection limit of a single copy ofHIV RNA. Infected cell frequencies measured with standard QVOA inHIV-infected subjects ART-treated in the chronic phase of the infectionis on the order of 0.1 to 1 infectious units per million (IUPM) of CD4 Tcell (PMID: 23459007) while mQVOA showed a median of 8.0 [3.2-11.6]IUPM.

We will use 50 million PBMCs for the mQVOA and will enrich PBMCs fortotal CD4+ T cells, expecting a recovery from 8 to 15 million persample. enriched CD4+ T cells will be serially diluted in a 24-wellplate coated with anti-CD3 and anti-CD28 monoclonal antibodies. We willperform 2-fold serial dilutions (4 dilutions). The startingconcentration will be 1×10⁶ cells/well and three replicates will beperformed depending on cell availability. After 2 days of stimulation,0.2×10⁶ MOLT-4/CCR5 cells will be added to each cell culture well (day0). Medium will be changed twice a week. Cell culture supernatants willbe collected at days 7 and 14 after the addition of MOLT-4/CCR5 cells.Cf-RNA from the supernatant (500 μl) will be extracted usingmagnetic-beads based technology, and quantified using a semi-nested realtime RT-PCR with a detection limit of a single copy of HIV RNA.Extracted viral RNA will be reverse transcribed and subjected to 16cycles of amplification with the following primers: Forward: 5′-ATG CCACGT AAG CGA AAC TCT GGG TCT CTC TDG TTA GAC-3′ (SEQ ID NO: 1); Reverse:5′-CCA TCT CTC TCC TTC TAG C-3′ (SEQ ID NO: 2). Pre-amplified productswill be diluted and subjected to a nested real time PCR for 40 cycles onthe Rotor-Gene Q by using the following primers and probes: Forward:5′-ATG CCA CGT AAG CGA AAC T-3′ (SEQ ID NO: 3); Reverse: 5′-CTG AGG GATCTC TAG TTA CC-3′ (SEQ ID NO: 4); Probe: 5′-LC-640-CAC TCA AGG CAA GCTTTA TTG AGG C-BBQ-3 (SEQ ID NO: 5). The number of wells positive for HIVRNA will be determined for each dilution and used to calculate the IUPMusing standard maximum likelihood methods.

HIV DNA measurements: We will measure the frequency of total CD4+ Tcells harboring total and integrated HIV DNA by using ourwell-established assays that can detect a single copy of the viralgenome in 10⁵ cells [8]. While integrated HIV DNA is present in bothlatently and productively infected cells, the ratio between total andintegrated has been recently shown to reflect residual HIV expressionand de-novo reverse transcription [17].

TILDA: We have developed a novel assay that measures the frequency ofcells harboring inducible HIV without the requirement of large bloodvolume [18]. By combining ultrasensitive detection of msRNAs uponstimulation together with a limiting dilution assay, this method allowsus to measure a frequency of cells harboring transcriptionallysilent—nonetheless inducible—viruses. To our knowledge, TILDA is theonly method that allows the measurement of the frequency of cellsharboring inducible virus that can be performed with less than a millionCD4⁺ T cells. Therefore, we will be able to measure the magnitude of thereservoir in sorted CD4⁺ T cells subsets at the leukapheresis timepoints, but also in total CD4+ T cells at several time points, includingthose at which the number of cells collected may be limited.

Schedule of Events

Screening (week −7 to −2): Informed consent is obtained, eligibility isdetermined.

Baseline assessments (week −2): Study subjects will undergo a largeblood draw of 120 mL (to determine a first baseline reservoir value bymQVOA).

Recombinant human growth hormone initiation (week 0: Subjects will besampled (large blood draw, 120 mL) before receiving the first dose ofSerostim® (3.0 mg/day) to generate a second baseline value by mQVOA.

Intermediate high dose (week 12): All subjects will receive safetyassessment including: symptom directed physical examination and safetylabs. A regular blood draw will be performed (30 mL).

Dose reduction (week 24): All enrolled subjects will be sampled (largeblood draw for an intermediate reservoir measurement by mQVOA) beforestarting the reduced dose regimen of recombinant human growthhormoneSerostim® (1.5 mg/day).

Intermediate low dose (week 36): All subjects will receive safetyassessment including: symptom directed physical examination and safetylabs. A regular blood draw will be performed (30 mL).

Study primary endpoint (week 48): Study subjects will undergo a largeblood draw (120 mL) to measure the reservoir by mQVOA. Serostim® therapyis stopped. End of the study.

REFERENCES

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We claim:
 1. A method of treating an HIV-1 infected subject comprisingthe administration of growth hormone (GH) to a subject infected withHIV-1.
 2. The method according to claim 1, said method furthercomprising the measuring the size of the replication competent HIVreservoir in said subject.
 3. The method according to claim 2, saidmethod comprising measuring the size of the replication competent HIVreservoir in said subject prior to the administration of GH to saidsubject and after the administration of GH to the subject.
 4. The methodaccording to claim 1, wherein GH is co-administered to a subject withone or more anti-retroviral drug (ARD).
 5. The method according to claim4, wherein said one or more anti-retroviral drug is selected fromAbacavir; Didanosine; Emtricitabine; Lamivudine; Stavudine; Tenofovirdisoproxil fumarate; Tenofovir alafenamide; Zalcitabine; Zidovudine;Delavirdine; Efavirenz; Etravirine; Nevirapine; Rilpivirine; Amprenavir;Atazanavir; Darunavir; Fosamprenavir; Indinavir; Lopinavir/Ritonavir;Nelfinavir; Ritonavir; Saquinavir; Tipranavir; Enfuvirtide; Maraviroc;Dolutegravir; Elvitegravir; Raltegravir; and combinations thereof.
 6. Amethod of treating an HIV-1 infected subject comprising theadministration of a first dose of growth hormone (GH) to a subjectinfected with HIV-1 for a first period of time followed by theadministration of a second dose of growth hormone (GH) to said subjectfor a second period of time.
 7. The method according to claim 6, saidmethod further comprising the measuring the size of the replicationcompetent HIV reservoir in said subject.
 8. The method according toclaim 7, said method comprising measuring the size of the replicationcompetent HIV reservoir in said subject prior to the administration ofsaid first dose of GH to said subject.
 9. The method according to claim7, said method comprising measuring the size of the replicationcompetent HIV reservoir in said subject after the administration of saidfirst dose of GH to said subject.
 10. The method according to claim 7,said method comprising measuring the size of the replication competentHIV reservoir in said subject prior to the administration of said firstdose of GH to said subject and at the conclusion of said first timeperiod.
 11. The method according to claim 7, said method comprisingmeasuring the size of the replication competent HIV reservoir in saidsubject prior to the administration of said first dose of GH to saidsubject, at the conclusion of said first period of time and at theconclusion of said second time period where said second dose of GH isadministered.
 12. The method according to claim 7, wherein the size ofthe replication competent HIV reservoir in said subject is measuredprior to the administration of said first dose of GH to said subject andat multiple time points after the administration of said first dose ofGH to the subject and multiple times after the administration of saidsecond dose of GH to the subject.
 13. The method according to claim 6,wherein said first and said second doses of GH are co-administered to asubject with one or more anti-retroviral drug (ARD).
 14. The methodaccording to claim 13, wherein said one or more anti-retroviral drug isselected from Abacavir; Didanosine; Emtricitabine; Lamivudine;Stavudine; Tenofovir disoproxil fumarate; Tenofovir alafenamide;Zalcitabine; Zidovudine; Delavirdine; Efavirenz; Etravirine; Nevirapine;Rilpivirine; Amprenavir; Atazanavir; Darunavir; Fosamprenavir;Indinavir; Lopinavir/Ritonavir; Nelfinavir; Ritonavir; Saquinavir;Tipranavir; Enfuvirtide; Maraviroc; Dolutegravir; Elvitegravir;Raltegravir; and combinations thereof.
 15. The method according to claim6, wherein said subject has not been treated with an ARD.
 16. The methodaccording to claim 6, wherein said subject is in an anti-retroviraltherapy (ART) free period.
 17. The method according to claim 13, whereinsaid subject has undergone ART for period of: at least 6 months; atleast 12 months; at least 18 months; or at least 24 months prior totreatment with GH.
 18. The method according to claim 6, wherein saidsecond dose is a reduced dose of GH.
 19. The method according to claim18, wherein said first dose of GH is administered in an amount of about2 mg/day to about 5 mg/day and said second dose is administered in anamount of about 1 mg/day to about 4 mg/day.
 20. The method according toclaim 19, wherein said first dose of GH is administered in an amount ofabout 3 mg/day and said second dose of GH is administered in an amountof about 1.5 mg/day.
 21. The method according to claim 6, wherein saidsubject is treated for a first period that ranges between about 2 weeksand about 52 weeks and a second period that ranges between about 2 andabout 52 weeks.
 22. The method according to claim 21, wherein said firstperiod is a period of about 24 weeks and said second period is a periodof about 24 weeks.
 23. The method according to claim 6, wherein saidsecond dose is an increased dose of GH.
 24. The method according toclaim 23, wherein said first dose of GH is administered in an amount ofabout 2 mg/day to about 5 mg/day and said second dose is administered inan amount of about 3.5 mg/day to about 25 mg/day.